Hyaluronan as a cytotoxic agent, drug pre-sensitizer and chemo-sensitizer in the treatment of disease

ABSTRACT

The present invention relates to the enhancement of bioavailability of chemotherapeutic agents for the treatment of disease. In particular the present invention relates to a method of enhancing the bioavailability of a chemotherapeutic agent comprising the step of administering to a subject in need thereof a therapeutically effective amount of hyaluronan.

FIELD OF THE INVENTION

[0001] The present invention relates to the enhancement ofbioavailability of chemotherapeutic agents for the treatment of disease.In particular the present invention relates to the use of hyaluronaneither alone or in combination with a chemotherapeutic agent toenhancement the bioavailability of the chemotherapeutic agent fortreatment of disease. The present invention also relates to thetreatment of a drug resistant disease whereby the drug resistance isovercome or alleviated with the use of hyaluronan either alone or incombination with a chemotherapeutic agent.

BACKGROUND TO THE INVENTION

[0002] Many diseases that afflict animals, including humans, are treatedwith chemotherapeutic agents. For example, chemotherapeutic agents haveproven valuable in the treatment of neoplastic disorders includingconnective or autoimmune diseases, metabolic disorders, anddermatological diseases, and many of these agents are highly effectiveand do not suffer from any bioavailability problems.

[0003] Proper use of chemotherapeutic agents requires a thoroughfamiliarity with the natural history and pathophysiology of the diseasebefore selecting the chemotherapeutic agent, determining a dose, andundertaking therapy. Each subject must be carefully evaluated, withattention directed toward factors which may potentiate toxicity, such asovert or occult infections, bleeding dyscrasias, poor nutritionalstatus, and severe metabolic disturbances. In addition, the functionalcondition of certain major organs, such as liver, kidneys, and bonemarrow, is extremely important. Therefore, the selection of theappropriate chemotherapeutic agent and devising an effective therapeuticregimen is influenced by the presentation of the subject. Suchconsiderations affect the dosage and type of drug administered.

[0004] Unfortunately, not all chemotherapeutics are readily useable. Forexample, some chemotherapeutic agents are inherently refractory in thatanimal cells do not readily respond to these agents, while otherchemotherapeutics suffer from acquired resistance. For instance, it iswell recognised that some subjects on prolonged chemotherapy are forcedto change chemotherapeutics as these become less efficacious with time.Moreover, some chemotherapeutics, while not affected by inherent oracquired resistance per se, are not effective in the treatment ofcertain diseases as they have innate problems with bioavailability. Onedisease that is frequently affected by both cellular resistance andbioavailability problems is cancer.

[0005] Cancer is responsible for one in four deaths in Western society.While the rates of new cases of cancer and deaths with cancer decreasedin the United States and Canada between 1990-1994, the data show that2,604,650 people in the United States died from cancer between1990-1994, with more men (53%) than women (47%) affected. The mostcommon cancer deaths were due to cancer of the lung (728,641), colon andrectum (285,724), breast (218,786), and prostate (169,943).

[0006] Among women, the most common cancers are breast (31%), lung(12%), colon and rectum (12%), uterus (6%), and ovary (4%), with breastand ovarian cancer representing approximately 35% of all cancers foundin women. The majority of women diagnosed with these forms of cancerreceive a combination of surgical, radiation therapy or chemotherapy.

[0007] Chemotherapeutic agents used to treat cancer can be subdividedinto several broad categories, including, (1) alkylating agents, such asmechlorethamine, cyclophosphamide, melphalan, uracil mustard,chlorambucil, busulfan, carmustine, lomustine, semustine,streptozoticin, and decrabazine; (2) antimetabolites, such asmethotrexate, fluorouracil, fluorodeoxyuridine, cytarabine, azarabine,idoxuridine, mercaptopurine, azathioprine, thioguanine, and adeninearabinoside; (3) natural product derivatives, such as vinblastine,vincristine, dactinomycin, daunorubicin, doxorubicin, mithramycin,taxanes (e. g., paclitaxel) bleomycin, etoposide, teniposide, andmitomycin C; and (4) miscellaneous agents, such as hydroxyurea,procarbezine, mititane, and cisplatinum.

[0008] Important cancer chemotherapeutic agents (with the usualeffective dosage) to which clinical multidrug-resistance has beenobserved include vinblastine (0.1 mg per kilogram per week), vincristine(0.01 mg per kilogram per week), etoposide (35 to 50 mg per square meterper day), dactinomycin (0.15 mg per kilogram per day), doxorubicin (500to 600 mg per square meter per week), daunorubicin (65 to 75 mg persquare meter per week), and mithramycin (0.025 mg per kilogram per day).

[0009] It is well appreciated by those skilled in the field that, atpresent, there are no effective means of overcoming cellular resistanceto chemotherapeutic agents. More importantly there are no practicalmeans of increasing bioavailability of chemotherapeutics withoutconcomitant increase in toxicity or side effects. Accordingly, there isa requirement for means of overcoming or at least alleviating theproblems associated with acquired or inherent cellular resistance aswell as means of increasing bioavailability of chemotherapeutics.

[0010] The applicant has previously investigated the usefulness ofhyaluronan (HA) as a drug delivery vehicle for chemotherapeutics, andfound that HA was useful when co-administered with these drugs.International patent application no. PCT/AU00/00004 was filed coveringthis invention, and is incorporated in its entirety herein by reference.HA, also known as hyaluronic acid, is a naturally occurringpolysaccharide comprising linear-chain polymers, which is foundubiquitously throughout the animal kingdom. HA is highly water-soluble,making it an ideal drug delivery vehicle for biological systems.

[0011] Subsequent to the filing of International patent application no.PCT/AU00/00004, the applicant surprising found that HA could act as asole agent. It was found that HA could exert a cytotoxic effect on humanbreast cancer cells, as well as pre-sensitizing cells so that theybecame more susceptible to chemotherapeutic agents. The presentinvention therefore provides methods whereby cells that were, or hadbecome resistant to chemotherapeutic agents could be effectivelytreated. More importantly, by using the disclosed methods it is possibleto decrease the dosages of chemotherapeutic agents without decreasingthe efficacy to the subject. The methods of the invention includeadministering hyaluronan either alone in conjunction with achemotherapeutic agent.

[0012] The present invention is based upon the discovery thathyaluronan, derivatives, analogues, and salts thereof, not only inhibitcells per se, but also allows the safe administration of selectedchemotherapeutic agents at standard or lower doses thought to be lesseffective, to treat subjects including human subjects. In vivoadministration of hyaluronan in combination with chemotherapeutic agentsalso enhances the therapeutic effect of these agents against cells thatare refractory, thus preventing the subsequent emergence of multidrugresistance.

[0013] Diseased cells such as cancer cells often have more permeablemembranes due to an alteration in the membrane potential, or increasedreceptor status which can alter the regulation of their intracellularmolecule transport which can result in cell swelling (Lang et al, 1993).While the applicant does not wish to be bound by any theory theypostulate that there are several mechanisms that could explain thecellular effect that HA is exerting both as a sole agent, and as apre-treatment for therapeutic agents:

[0014] 1). When HA is bound to CD44, RHAMM and the scavenger receptorbound, the nett negative charge of HA alters the membrane potential ofthe cell resulting in an increase in cell permeability consequentlyenabling a greater flux of drug into the diseases cell.

[0015] 2). When HA is bound to diseased cells such as tumour cells andinternalised there could be a hyperosmotic effect resulting in celllysis.

[0016] 3). HA could exert oxidative membrane damage resulting inapoptosis.

[0017] 4). HA internalisation could elevate the mitochondrial membranepotential which could result in cell death or increased drug retention.

[0018] Since HA is administered at satuarable levels, there would be aconstant internalisation of the glycosaminoglycan which means that anytherapeutic agent which is in an equilibrium within the volumetricdomain of the HA is co-internalised resulting in a concentratedintracellular release of the drug

SUMMARY OF INVENTION

[0019] In its broadest aspect the present invention provides a method oftreating a subject in need thereof comprising the step of administeringto said subject a therapeutically effective amount of hyaluronan inconjunction with a chemotherapeutic agent such that saidchemotherapeutic agent is more effective than when administered alone.

[0020] The present invention also provides a method of enhancing thebioavailability of a chemotherapeutic agent comprising the step ofadministering to a subject in need thereof a therapeutically effectiveamount of hyaluronan.

[0021] Hyaluronan can be used to significantly enhance thebioavailability of any administered chemotherapeutic agent. Preferably,the chemotherapeutic agent that is administered is selected from thegroup consisting of carmustine (BCNU), chlorambucil (Leukeran),cisplatin (Platinol), Cytarabine, doxorubicin (Adriamycin), fluorouracil(5-FU), methoxetrate (Mexate), CPT111, etoposide, plicamycin (Mithracin)and taxanes such as, for example, paclitaxel.

[0022] In yet another embodiment, the invention provides a method oftreating or preventing multidrug resistance or drug-resistant cellscomprising the step of administering a therapeutically effective amountof hyaluronan, prior to, together with, or subsequent to theadministration of a chemotherapeutic agent.

[0023] As described more fully below, administration of hyaluronan and achemotherapeutic agent results in the suppression of tumor growth by atleast 50%; preferably 60%; and, more preferably, greater than 70%.Accordingly, the elimination of tumor growth and proliferationeliminates the production of multidrug resistant cells reducing therecurrence of cancer and increasing the efficacy of chemotherapeutictreatments.

[0024] The present invention further provides a pharmaceuticalcomposition for increasing the sensitivity of cells to chemotherapeuticagents comprising hyaluronan. The hyaluronan and/or chemotherapeuticagent may also be administered together with a further pharmaceuticalcarrier.

[0025] The present invention also provides a method of treating cancercells comprising the step of administering to a patient in thereof atherapeutically effective amount of hyaluronan.

[0026] Typically said cancer cells are resistant to chemotherapeuticdrugs.

[0027] In a further aspect of the present invention there is provided amethod of overcoming cellular resistance, comprising the step ofadministering a therapeutically effective amount of HA.

[0028] Throughout the description and claims of this specification, theword “comprise” and variations of the word, such as “comprising” and“comprises”, means “including but not limited to” and is not intended toexclude other additives, components, integers or steps.

BRIEF DESCRIPTION OF THE FIGURES

[0029]FIG. 1 shows exponentially growing breast cancer cells exposed to750,000 dalton HA for 24 h at which stage the cells were photographed.At 10 ng/ml there was a reduction in cell number, but no difference inmorphology was noted. At 100 ng/ml and 1 μg/ml the cells appeared top beundergoing a osmotic response where the cells appeared to swell. At 2mg/ml and 5 mg/ml the cells became granular and the plasma membrane was“pitted” possibly indicating an osmotic response and/or the commencementof cell death.

[0030]FIGS. 2a-2 f shows exponentially growing breast cancer cells thatwere exposed to 750,000 dalton HA for 30 min, 1 h, or 24 h at whichstage the cells were varying concentrations of adriamycin. These figuresalso illustrate the effect of HA/drug co-incubation for the period of 1or 3 days. These diagrams illustrate that HA can “pre-sensitise” and/orchemosensitise cells to therapeutic drugs.

[0031]FIGS. 3a-3 d shows exponentially growing breast cancer cellsexposed to varying concentrations of 750,000 dalton hyaluronan for 1 h,24 h or 3 days followed by treatment with 40 nM Adriamycin for varyingtime periods of 1 h, 24 h or 3 days. These figures show that a wideconcentration range of hyaluronan can act as a chemosenitiser or exert acytotoxic effect.

[0032]FIG. 4 shows that there was no treatment toxicity noted throughoutthe 6-week study. In comparison to the 5-FU treatment group the micereceiving HA therapy, that is as a sole agent or as a chemosensitizer,demonstrated enhanced well being where the animal did not loose weight,but maintained its body mass.

[0033]FIG. 5 shows that at the end of the 6 week study, tumour mass wasdetermined where the HA chemosensitizing therapy had significantlysmaller tumours than the saline group, HA and 5-FU groups (p=0.005). HAas a sole agent also demonstrated its effect by reducing the primarytumour mass in comparison to the saline control. No significantdifferences in tumour response were noted in the initial 2 weeks oftreatment, but thereafter the HA followed by 5-FU tumour growth wasretarded in comparison to the other treatment groups. During the 6 weeksof treatment interesting differences were noted in the number of tumourdoubling cycles. Mice receiving the saline treatment underwent anaverage of 4 tumour doublings, while the incorporation of HA into thetreatment regimen significantly increased the tumour doubling time whereHA/5-FU animals underwent an average of one tumour doubling cycle, onceagain highlighting the effect of HA on 5-FU cytotoxicity.

[0034]FIG. 6 shows that the co-administration of HA resulted in asignificant reduction in non-lymphoid metastasis. With the exception ofthe mice receiving the HA therapy, new tumours were observed around theneck or underarm region of the area adjacent to the primary tumour.

DETAILED DESCRIPTION OF THE INVENTION

[0035] The methods and compositions of the invention are useful forincreasing the sensitivity of cells to chemotherapeutic agents such as,for example, anti-cancer agents like paclitaxel, analgesics, opiates,hormones or antibiotics and the like. In particular the methods andcompositions of the invention are useful for increasing the sensitivityof cells associated with cellular proliferative disorders (eg., aneoplasm). By increasing the efficacy without concomitant toxicity tonon-cancer cells the invention provides methods and compositions usefulfor treating tumors and preventing or reducing the chances of relapseand death as a result of cytotoxicity. In addition, the inventioneliminates or reduces the number of multidrug resistant cells byeliminating cancer cells prior to any mutation inducing a multidrugresistant phenotype. Accordingly, by reducing multi-drug resistant tumorcells from arising, the invention satisfies the shortcomings of currenttherapeutic modalities.

[0036] The term “subject” as used herein refers to any animal having adisease or condition which requires treatment with a chemotherapeuticagent wherein the chemotherapeutic agent has reduced efficacy relativeto that desired. Preferably the subject is suffering from a cellularproliferative disorder (eg., a neoplastic disorder). Subjects for thepurposes of the invention include, but are not limited to, mammals (eg.,bovine, canine, equine, feline, porcine) and preferably humans.

[0037] By “cell proliferative disorder” is meant that a cell or cellsdemonstrate abnormal growth, typically aberrant growth, leading to aneoplasm, tumor or a cancer.

[0038] Cell proliferative disorders include, for example, cancers of thebreast, lung, prostate, kidney, skin, neural, ovary, uterus, liver,pancreas, epithelial, gastric, intestinal, exocrine, endocrine,lymphatic, haematopoietic system or head and neck tissue.

[0039] Generally, neoplastic diseases are conditions in which abnormalproliferation of cells results in a mass of tissue called a neoplasm ortumor. Neoplasms have varying degrees of abnormalities in structure andbehaviour. Some neoplasms are benign while others are malignant orcancerous. An effective treatment of neoplastic disease would beconsidered a valuable contribution to the search for cancer preventiveor curative procedures.

[0040] The methods of this invention involve in one embodiment, (1) theadministration of hyaluronan, prior to, together with, or subsequent tothe administration of a chemotherapeutic agent; or (2) theadministration of a combination of hyaluronan and a chemotherapeuticagent.

[0041] As used herein, the term “therapeutically effective amount” ismeant an amount of a compound of the present invention effective toyield a desired therapeutic response. For example to prevent cancer ortreat the symptoms of cancer in a host or an amount effective to treatcancer.

[0042] The specific “therapeutically effective amount” will, obviously,vary with such factors as the particular condition being treated, thephysical condition of the patient, the type of mammal being treated, theduration of the treatment, the nature of concurrent therapy (if any),and the specific formulations employed and the structure of thecompounds or its derivatives.

[0043] As used herein, a “pharmaceutical carrier” is a pharmaceuticallyacceptable solvent, suspending agent or vehicle for delivering thehyaluronan and/or chemotherapeutic agent to the animal or human. Thecarrier may be liquid or solid and is selected with the planned mannerof administration in mind.

[0044] As used herein, “cancer” refers to all types of cancers orneoplasm or malignant tumours found in mammals. Cancer includessarcomas, lymphomas and other cancers. The following types are examples,but are, but is not intended to be limited to these particular types ofcancers: prostate, colon, breast, both the MX-1 and the MCF lines,pancreatic, neuroblastoma, rhabdomysarcoma, home, lung, murine,melanoma, leukemia, pancreatic, melanoma, ovarian, brain, head & neck,kidney, mesothelioma, sarcoma, Kaposi's, sarcoma, stomach, and uterine.

[0045] As used herein, the term “cell” include but is not limited tomammalian cells (eg., mouse cells rat cells or human cells).

[0046] The hyaluronan and/or chemotherapeutic agents may be administeredorally, topically, or parenterally in dosage unit formulationscontaining conventional non-toxic pharmaceutically acceptable carriers,adjuvants, and vehicles. The term parenteral as used herein includessubcutaneous injections, aerosol, intravenous, intramuscular,intrathecal, intracranial, intrasternal injection or infusiontechniques.

[0047] The present invention also provides suitable topical, oral, andparenteral pharmaceutical formulations for use in the novel methods oftreatment of the present invention. The compounds of the presentinvention may be administered orally as tablets, aqueous or oilysuspensions, lozenges, troches, powders, granules, emulsions, capsules,syrups or elixirs. The composition for oral use may contain one or moreagents selected from the group of sweetening agents, flavouring agents,colouring agents and preserving agents in order to producepharmaceutically elegant and palatable preparations. The tablets containthe active ingredient in admixture with non-toxic pharmaceuticallyacceptable excipients which are suitable for the manufacture of tablets.

[0048] These excipients may be, for example, (1) inert diluents, such ascalcium carbonate, lactose, calcium phosphate or sodium phosphate; (2)granulating and disintegrating agents, such as corn starch or alginicacid; (3) binding agents, such as starch, gelatin or acacia; and (4)lubricating agents, such as magnesium stearate, stearic acid or talc.These tablets may be uncoated or coated by known techniques to delaydisintegration and absorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period. For example, a timedelay material such as glyceryl monostearate or glyceryl distearate maybe employed. Coating may also be performed using techniques described inthe U.S. Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmotictherapeutic tablets for control release.

[0049] The hyaluronan as well as the chemotherapeutic agents useful inthe method of the invention can be administered, for in vivoapplication, parenterally by injection or by gradual perfusion over timeindependently or together. Administration may be intravenously,intraperitoneally, intramuscularly, subcutaneously, intracavity, ortransdermally. For in vitro studies the agents may be added or dissolvedin an appropriate biologically acceptable buffer and added to a cell ortissue.

[0050] Preparations for parenteral administration include sterileaqueous or non-aqueous solutions, suspensions, and emulsions. Examplesof non-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's intravenousvehicles include fluid and nutrient replenishers, electrolytereplenishers (such as those based on Ringer's dextrose), and the like.Preservatives and other additives may also be present such as, forexample, anti-microbials, anti-oxidants, chelating agents, growthfactors and inert gases and the like.

[0051] It is envisioned that the invention can be used to treatpathologies associated cell proliferative disorders, including, forexample, neoplasms, cancers (eg., cancers of the breast, lung, prostate,kidney, skin, neural, ovary, uterus, liver, pancreas, epithelial,gastric, intestinal, exocrine, endocrine, lymphatic, haematopoieticsystem or head and neck tissue), fibrotic disorders and the like.

[0052] The methods and compounds of the invention may also be used totreat other diseases associated with chemotherapeutic treatment such asneurodegenerative disorders, hormonal imbalance and the like. Therefore,the present invention encompasses methods for ameliorating a disorderassociated with cell proliferation, neoplasms, cancers and the like,including treating a subject having the disorder, at the site of thedisorder, with hyaluronan and a chemotherapeutic agent in an amountsufficient to inhibit or ameliorate the cell's proliferation or thedisorder. Generally, the terms “treating”, “treatment” and the like areused herein to mean affecting a subject, tissue or cell to obtain adesired pharmacologic and/or physiologic effect. The effect may beprophylactic in terms of completely or partially preventing a cellproliferative disorder or sign or symptom thereof, and/or may betherapeutic in terms of a partial or complete cure for a disorder and/oradverse effect attributable to, for example, aberrant cellproliferation. “Treating” as used herein covers any treatment of, orprevention of a cell proliferative disorder in a vertebrate, a mammal,particularly a human, and includes: (a) preventing the disorder fromoccurring in a subject that may be predisposed to the disorder, but hasnot yet been diagnosed as having it; (b) inhibiting the disorder, i.e.,arresting its development; or (c) relieving or ameliorating thedisorder, i. e., cause regression of the disorder.

[0053] The invention includes various pharmaceutical compositions usefulfor ameliorating cell proliferative disorder, including neoplasms,cancers and the like. The pharmaceutical compositions according to oneembodiment of the invention are prepared by bringing hyaluronan,analogue, derivatives or salts thereof and one or more chemotherapeuticagents or combinations of hyaluronan and one or more chemotherapeuticagents into a form suitable for administration to a subject usingcarriers, excipients and additives or auxiliaries. Frequently usedcarriers or auxiliaries include magnesium carbonate, titanium dioxide,lactose, mannitol and other sugars, talc, milk protein, gelatin, starch,vitamins, cellulose and its derivatives, animal and vegetable oils,polyethylene glycols and solvents, such as sterile water, alcohols,glycerol and polyhydric alcohols. Intravenous vehicles include fluid andnutrient replenishers. Preservatives include antimicrobial,anti-oxidants, chelating agents and inert gases. Other pharmaceuticallyacceptable carriers include aqueous solutions, non-toxic excipients,including salts, preservatives, buffers and the like, as described, forinstance, in Remington's Pharmaceutical Sciences, 15th ed. Easton: MackPublishing Co., 1405-1412,1461-1487 (1975) and The National FormularyXIV., 14th ed. Washington: American Pharmaceutical Association (1975),the contents of which are hereby incorporated by reference. The pH andexact concentration of the various components of the pharmaceuticalcomposition are adjusted according to routine skills in the art. SeeGoodman and Gilman's The Pharmacological Basis for Therapeutics (7thed.).

[0054] The pharmaceutical compositions are preferably prepared andadministered in dose units. Solid dose units are tablets, capsules andsuppositories. For treatment of a subject, depending on activity of thecompound, manner of administration, nature and severity of the disorder,age and body weight of the subject, different daily doses can be used.Under certain circumstances, however, higher or lower daily doses may beappropriate. The administration of the daily dose can be carried outboth by single administration in the form of an individual dose unit orelse several smaller dose units and also by multiple administration ofsubdivided doses at specific intervals.

[0055] The pharmaceutical compositions according to the invention may beadministered locally or systemically in a therapeutically effectivedose. Amounts effective for this use will, of course, depend on theseverity of the disease and the weight and general state of the subject.Typically, dosages used in vitro may provide useful guidance in theamounts useful for in situ administration of the pharmaceuticalcomposition, and animal models may be used to determine effectivedosages for treatment of particular disorders. Various considerationsare described, eg., in Langer, Science, 249: 1527, (1990). Formulationsfor oral use may be in the form of hard gelatin capsules wherein theactive ingredient is mixed with an inert solid diluent, for example,calcium carbonate, calcium phosphate or kaolin. They may also be in theform of soft gelatin capsules wherein the active ingredient is mixedwith water or an oil medium, such as peanut oil, liquid paraffin orolive oil.

[0056] Aqueous suspensions normally contain the active materials inadmixture with excipients suitable for the manufacture of aqueoussuspension. Such excipients may be (1) suspending agent such as sodiumcarboxymethyl cellulose, methyl cellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;(2) dispersing or wetting agents which may be (a) naturally occurringphosphatide such as lecithin; (b) a condensation product of an alkyleneoxide with a fatty acid, for example, polyoxyethylene stearate; (c) acondensation product of ethylene oxide with a long chain aliphaticalcohol, for example, heptadecaethylenoxycetanol; (d) a condensationproduct of ethylene oxide with a partial ester derived from a fatty acidand hexitol such as polyoxyethylene sorbitol monooleate, or (e) acondensation product of ethylene oxide with a partial ester derived fromfatty acids and hexitol anhydrides, for example polyoxyethylene sorbitanmonooleate.

[0057] The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleagenous suspension. This suspension may beformulated according to known methods using those suitable dispersing orwetting agents and suspending agents which have been mentioned above.The sterile injectable preparation may also a sterile injectablesolution or suspension in a non-toxic parenterally-acceptable diluent orsolvent, for example, as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution, and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose, any bland fixed oil may be employedincluding synthetic mono-or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

[0058] Hyaluronan together with a chemotherapeutic agent of the presentinvention may also be administered in the form of liposome deliverysystems, such as small unilamellar vesicles, large unilamellar vesicles,and multilamellar vesicles. Liposomes can be formed from a variety ofphospholipids, such as cholesterol, stearylamine, orphosphatidylcholines.

[0059] Dosage levels of the compounds of the present invention are ofthe order of about 0.5 mg to about 10 mg per kilogram body weight, witha preferred dosage range between about 5 mg to about 20 mg per kilogrambody weight per day (from about 0.3 gms to about 1.2 gms per patient perday). The amount of active ingredient that may be combined with thecarrier materials to produce a single dosage will vary depending uponthe host treated and the particular mode of administration. For example,a formulation intended for oral administration to humans may containabout 5 mg to 1 g of an active compound with an appropriate andconvenient amount of carrier material which may vary from about 5 to 95percent of the total composition. Dosage unit forms will generallycontain between from about 5 mg to 500 mg of active ingredient.

[0060] It will be understood, however, that the specific dose level forany particular patient will depend upon a variety of factors includingthe activity of the specific compound employed, the age, body weight,general health, sex, diet, time of administration, route ofadministration, rate of excretion, drug combination and the severity ofthe particular disease undergoing therapy.

[0061] In addition, some of the compounds of the instant invention mayform solvates with water or common organic solvents. Such solvates areencompassed within the scope of the invention.

[0062] The compounds of the present invention may additionally becombined with other compounds to provide an operative combination. It isintended to include any chemically compatible combination ofchemotherapeutic agents, as long as the combination does not eliminatethe activity of the hyaluronan of this invention.

[0063] The invention will now be further described by way of referenceonly to the following non-limiting examples. It should be understood,however, that the examples following are illustrative only, and shouldnot be taken in any way as a restriction on the generality of theinvention described above. In particular, while the invention isdescribed in detail in relation to cancer, it will be clearly understoodthat the findings herein are not limited to treatment of cancer. Forexample, HA may be used for treatment of other conditions.

EXAMPLE 1 Preparation of Hyaluronan and 5-Fluorouracil Solutions

[0064] HA used in all of the in vitro and in vivo studies were obtainedfrom Kyowa Hakko Kogyo (Yamaguchi, Japan). 5-FU was obtained from Sigma,St. Louis, USA. And Adraimycin from Cytomix, Northcote, Melbourne,Australia. A standard profile of the HA used is shown in Table 1. TABLE1 Specification Sheet For Hyaluronan Bulk Dried Powder TESTSPECIFICATION 1. Description White or cream coloured powder or granules,odourless 2. Identification (IR Spectrum) Conforms to Reference Standard3. pH (1% solution) 5.0 to 7.0 4. Loss on Drying NMT 10.0% 5. Residue onIgnition 15.0 to 19.0% 6. Protein Content NMT 0.1% 7. Heavy Metals NMT20 ppm 8. Arsenic NMT 2 ppm 9. Sodium Hyaluronate Assay 97.0-102.0%(dried basis) 10. Intrinsic Viscosity 10.0-14.5 dL/g 11. Total AerobicMicrobial Count NMT 50 CFU/gram (USP 23) 12. Staphylococcus aureus (USP23) Absent 13. Pseudomonas aeruginosa (USP 23) Absent 14. Yeasts andMoulds (USP 23) NMT 50 CFU/gram 15. Bacterial Endotoxin NMT 0.07 EU/mg(LAL) (USP23)

[0065] A 10 mg/ml stock of HA solution was prepared by dissolvingdesiccated HA (modal M_(r) 7.5×10⁵ kDa,) in pyrogen-free injection gradewater. To ensure a homogenous solution the HA was dissolved overnight at4° C. followed by thorough vortexing. To ensure that the HA hadmaintained its molecular weight during the preparation of the stocksolution, the solution was analysed on a Sephacryl S-1000 size exclusiongel with column specifications of 1.6 cm×70 cm, sample size 2 ml, flowrate 18 ml/h and 2 ml fraction size. Hyaluronan was detected in columnfractions by the uronic acid assay.

[0066] The uronic acid assay was used to detect the presence ofhyaluronan qualitatively from the fractions collected from the gelfiltration chromatography procedure. A 25 μl aliquot of each fractionwas then transferred into a 96 well plate. 250 μl of a carbazole reagent(3M carbazole/0.025M borate in H₂SO₄) was then added to these fractions.The 96 well plate was incubated for 45-60 min at 80° C. A DynatechMR7000 plate reader with a 550 nm filter was used to read the 96 wellplate. The absorbance was considered to be significant when it was >3standard deviations above the background absorbance. The background wascalculated by taking an equal number of sample points before and afterV_(o) and V_(t) where the average number taken was 16 (Fraser et al.1998).

[0067] A stock solution of 5-FU was prepared by dissolving powdered 5-FUin 0.1M NaOH (pH 8.9) and brought to a concentration of 1 mg/ml withpyrogen-free injection grade 0.9% w/v NaCl. The stock solution wasfiltered through a 0.22 μm filter to ensure sterility. The 5-FU wasdiluted by adding the required volume of stock solution to the cell-linespecific growth medium as specified above.

[0068] A 10 mg/ml solution of adriamycin in 0.9% NaCl was obtained fromCytomix.

EXAMPLE 2 Testing the Effect of Hyaluronan on Cancer Cell Morphology

[0069] Human breast adenocarcinoma cell lines MDA-MB-468, MDA-MB-435 andMDA-MB-231 were selected based on HA binding affinity (Culty et al,1994), and the expression of the HA receptors of CD44 and RHAMM (Wang etal, 1996). The characteristics of these cell lines are shown in Table 2.TABLE 2 Hyaluronan Binding And Receptor Expression Of Human MammaryCarcinoma Cell Lines HA Receptor Type of breast Degree of HAExpression^(b) Cell Line cancer Binding^(a) CD44 RHAMM MDA-MB-231adenocarcinoma ++ +++ +++ MDA-MB-468 adenocarcinoma ++++ ++++ ++MDA-MB-435 ductal + +++ ND carcinoma

[0070] Cell lines MDA-MB-468, MDA-MB-435 and MDA-MB-231 were routinelygrown and subcultured as a monolayer in 175 cm² culture flasks inLeibovitz L-15 Medium supplemented with 10% Foetal calf serum (FCS) andantibiotic/antimycotic reagents at 37° C. in humidity controlledincubator with 100% (v/v) air.

[0071] Leibovitz-L-15 with glutamine (10× concentrate), RPMI (10×concentrate), Eagles basal medium (EBM, 10× concentrate), 20 mM HEPES,0.09% w/v bicarbonate, Hanks' Balanced Salt Solution (HBSS, 10×concentrate) and Dulbecco's Phosphate Buffered Saline without calciumand magnesium (PBS, 10× concentrate) were purchased from Sigma (StLouis, Mo., USA). Powder concentrates were dissolved in the requiredvolume of reverse osmosis deionised pyrogen-free distilled water to makea single strength solution, sterilised by 0.22 μm high pressurefiltration (Millipore Corporation, MA. U.S.A.), and stored at 4° C. FCS)were purchased from the CSL Ltd., Australia. FCS was stored at −20° C.Antibiotic/antimycotic solution (100× concentrate) containing 10,000units penicillin, 10 mg streptomycin and 25 μg amphotericin U/ml wasobtained from Sigma (St Louis, USA). Trypsin/EDTA solution (10×concentrate) containing 5 g porcine trypsin and 2 g EDTA/L in 0.9% w/vsodium chloride was obtained from Sigma (St Louis, Mo., USA). All breastcancer cell lines were purchased from American tissue culture collection(Rockville, USA). All plastic disposable culture vessels were purchasedfrom Greiner (Austria). Eight-welled, tissue culture microscope slideswere obtained from Linbro (Flow Laboratories, VA, USA).

[0072] For the tests, MDA MB-468, MDA MB-231 and MDA MB-435 cell linewere grown in 90% Leibovitz L-15 medium supplemented with 10% FCS. Whenconfluent the cultures were washed 1× in HBSS and trypsinised in 0.25%trypsin/0.05% EDTA. The cell suspensions were counted with an automatedcell counter (ZM-2 Coulter Counter) by adding 15 mL saline+0.2 ml ofcell suspension.

[0073] Cells were resuspended to a number of:

[0074] MDA MB-468: 25,000 cell/ml of media

[0075] MDA MB-231: 12,000 cell/ml of media

[0076] MDA MB-435: 12,000 cell/ml of media

[0077] The cells were plated into 48-well plates (1 cm² surface area) byadding 1 ml of cell suspension per well. Cells were allowed to attachfor 24 h, before the media was removed, monolayers washed. The testmedia was; growth media containing 0-1 μm adriamycin or 5-fluorouracilwith or without the addition of 0-1 μM of HA (modal Mw 750,000). Thecells were exposed to the several combinations of HA and drugs fordifferent times and at different concentrations (Table 3). TABLE 3Incubation Conditions for Hyaluronan and Drugs with Human Breast CancerCells HA Drug Incubation Incubation Growth Sequence of HA/Drug AdditionTime Time Time 0-1 μM HA, media wash, 0-1 μM 30 min  1 h 1 day drug,media wash, grow drug- free 0-1 μM HA, media wash, 0-1 μM  1 h  1 h 1day drug, media wash, grow drug- free 0-1 μM HA, media wash, 0-1 μM 24 h 1 h 1 day drug, media wash, grow drug- free 0-1 μM HA, media wash, 0-1μM 24 h 24 1 day drug, media wash, grow drug- free 0-1 μM HA, mediawash, 0-1 μM 30 min  1 h 3 day drug, media wash, grow drug- free 0-1 μMHA, media wash, 0-1 μM  1 h  1 h 3 day drug, media wash, grow drug- free0-1 μM HA, media wash, 0-1 μM 24 h  1 h 3 day drug, media wash, growdrug- free 0-1 μM HA, media wash, 0-1 μM 24 h 24 3 day drug, media wash,grow drug- free 0-1 μM drug/100 nM HA 30 min 1 day 0-1 μM drug/100 nM HA 1 h 1 day 0-1 μM drug/100 nM HA 24 1 day 0-1 μM drug/100 nM HA 30 min 3days 0-1 μM drug/100 nM HA  1 h 3 days 0-1 μM drug/100 nM HA 24 3 days0-1 μM HA 30 min 1 day 0-1 μM HA  1 h 1 day 0-1 μM HA 24 1 day 0-1 μM HA30 min 3 days 0-1 μM HA  1 h 3 days 0-1 μM HA 24 3 days 0-1 μM HA 3 days3 days

[0078] After the incubation and growth periods the cell monolayers werewashed with HBSS and trypsinised in 0.25% trypsin/0.05% EDTA. The cellsuspensions were counted with an automated cell counter (ZM-2 CoulterCounter) by adding 15 mL saline+0.2 ml of cell suspension. Results wereexpressed as % of no drug control which was calculated as:$\frac{{Cell}\quad {count} \times 100}{{Cells}\quad {in}\quad {no}\quad {drug}\quad {control}}$

[0079] Or depending on the experiment as % of drug control, calculatedas:$\frac{{Cell}\quad {count} \times 100}{{Cells}\quad {in}\quad {drug}\quad {control}}$

[0080] Exponentially growing human breast cancer cells MDA MB 231 asdescribed in example 2 were incubated with 0-5 mg/ml HA (modal Mr750,000D) for 24 h. At 24 h the cells were counted and photographed withCPR, 1600 film rolls from Eastman Kodak Company, Rochester, USA.

[0081] When HA was incubated with breast cancer cells for 30 min, 1 h,24 h or 3 days a varied response was observed, where the reduction inbreast cancer cell number ranged from 0-29% (See Table 4). TABLE 4Cytotoxic Effect of HA on Human Breast Cancer Cell Lines Exposure CellLine Cell Line Cell Line Time MDA-MB 468 MDA-MB 231 MDA-MB 435 3 days−29%  −23% −22% 100 nM 1 h 100 nM +3% −21%  −4% 30 min −5% −27% −12% 100nM 30 min −22%  0 500 nM 30 min +2% −26% ND 1000 nM 24 h 100 nM −5%  −8%−12%

[0082] When human breast cancer cells were incubated with HA specificmorphological changes (See FIG. 1) were also observed such as swellingof the plasma membrane, greater granularity of cytosolic components.

[0083] When human breast cancer cells were exposed to HA for 30 min, 1h, 24 h or 3 days followed by exposure toadriamycin, it became evidentthat HA ehanced the cytotoxicity of the drug (FIG. 3 & Table 5). TABLE 5Effect of HA on Adriamycin Cytotoxicity in Breast Cancer Cell Lines IC₅₀IC₅₀ IC₅₀ Treatment MDA-MB 468 MDA-MB 231 MDA-MB 435 3 day drug 3 to 124 to 5 10 exposure 1 h drug/HA, 3 40 2 to 8  0 days drug-free 1 h drug,3 days 20 to 40  3 to 9  6 to 10 drug-free 30 min 100 nm HA, 2 to 20 2to 6  4 to 40 1 hr drug, 3 days drug-free 30 min 100 nM HA, 3 to 18 2 to4 2 to 8 3 day drug exposure 30 min 500 nM HA, 3 to 9  2 to 8 2 to 4 3day drug exposure 30 min 1000 nM HA, 1 to 10 2 to 8 1 to 5 3 day drugexposure 24 h 100 nM HA, 3 8 to 12 13 24 day drug exposure 24 h 100 nMHA, 1h 50 to 60   9 21 drug exposure, drug-free 3 days

[0084] All figures represent the range of 2-3 separate experiments,where the numerical values are the multiplication factor decrease inIC₅₀ which is exerted by the addition of HA to drug or pre-sensitizationof cancer cells with HA before the addition of drug.

EXAMPLE 3 Efficacy of Hyaluronan In Vivo

[0085] Based on the results from the in vitro drug sensitivityexperiments in Example 2, evaluation of the treatment efficacy ofhyaluronan as a sole agent, and as a chemosensitizer in the treatmenthuman breast carcinomas in vivo was undertaken.

[0086] From the results in Example 2 the carcinoma cell line MDA-MB-468was selected as the cancer cell inoculant for the generation of any nudemouse human tumour xenografts. Cells were routinely grown andsubcultured as a previously described in Example 2. For injection intomice, cells were grown to 100% confluency, trypsinised in 0.025%trypsin/0.01% EDTA solution, washed twice by centrifugation in a BeckmanTJ-6 bench centrifuge at 400 g_(av) for 10 min, counted using a Model-ZMCoulter counter and resuspended in serum-free Leibovitz L-15 medium at1×10⁸ cells/ml.

[0087] Six to eight weeks old athymic CBA/WEHI nude female mice,purchased from the Walter and Eliza Hall Research Institute, MelbourneAustralia, were maintained under specific pathogen-free conditions, withsterilised food and water available ad libitum. Each mouse received oneinjection containing 5×10⁶ cells in 50 μl. The cells were injected witha 26 gauge needle into the mammary fat pad directly under the firstnipple (Lamszus et al, 1997). Tumour measurements were made weekly bymeasuring three perpendicular diameters (d₁d₂d₃). Tumour volume wasestimated using the formula:

(⅙)π(d₁d₂d₃)

[0088] Treatment with 5-FU±HA was commenced approximately 4-8 weeksafter the cancer cell inoculation. The mean tumour size for mice used ineach study is summarised in Table 6. TABLE 6 Summary of Human BreastCancer Tumours at Commencement of Each Study Tumour volume Tumour as %of net body Study (mean ± SEM) mass (mean ± SEM) Efficacy: 6- 0.37 ±0.20 mm³ 0.19 ± 0.10 mm³ week

[0089] Approximately 8 weeks after tumour induction two tumour-bearingmice were given a lethal dose of Nembutal. Within 3 min of killing themice, tumours were surgically removed and immediately fixed in 10%buffered formalin for 12 h. The fixed tumour was dehydrated overnight ina series of 70-100% ethanol, followed by paraffin embedding from which2-4 μm sections were cut. The sections were placed on slides, de-waxed,and brought to water. Slides were washed 3×5 min in PBS. Heterophileproteins were blocked by incubation with 10% foetal calf serum for 10min, followed by a PBS rinse.

[0090] Secondary antibodies used in the visualisation of HA and HAsynthase antibodies were purchased from Dako (California, U.S.A.).3,3′-Diaminobenzidine (Sigma Fast DAB) tablets were obtained from Sigma,St. Louis, USA.

[0091] The detection antibodies were applied for 60 min at RT. Thedetection antisera or antibodies were against RHAMM , CD44H and CAE. Theslides were washed 3×5 min in PBS and endogenous peroxidase activityblocked by immersion in 0.3% H₂O₂ in methanol for 20 min. Following afurther PBS wash, the peroxidase-conjugated swine anti-rabbit secondaryantiserum was applied for 60 min at RT, followed by 3×5 min washes inPBS. Sigma Fast 3,3′-Diaminobenzidine tablets (DAB) were preparedaccording to the manufacturer's instructions and the DAB solution wasapplied for 5-10 min at RT. The slides were washed in tap water for 10min, counterstained with haematoxylin, dehydrated and mounted.

[0092] Individual injections of 5-FU were prepared according toindividual mouse masses, with the aim of delivering 30 mg/kg 5-FU in 50μl (equivalent to human therapeutic dose of 10.5 mg/kg for a mean bodyweight of 60 kg; Inaba et al, 1988). HA injection comprising a final HAconcentration equivalent to 12.5 mg/kg of mouse mass were prepared sothat deliver of 12.5 mg/kg HA in 50 μl could be effected. With thisquantity of HA injected into the body, saturation kinetics would beobserved for the period of the experimentation (Fraser et al, 1983).

[0093] One of the most commonly used treatment regimens for human breastcancer is cyclophosphamide, methotrexate and 5-fluorouacil, which isadministered on day 1 and 8 of a 28 day cycle. In human breast cancerthe initial treatment regimen is for 6 cycles at which time the patientcondition is re-assessed, therefore we tried to simulate the humantreatment regimen as closely as possible by exposing the mice to 6cycles (6 months) of treatment in a long term efficacy study and a 6cycles (6 week) short term efficacy study. Considering the life cycle ofa mouse is approximately 2 years we commenced both short-term andlong-term treatment protocols (see Table 7). TABLE 7 TreatmentAdministration Protocols. 6-Week Study Treatment Regimen Bolus TreatmentGroup Dosage injection on Days 1. Saline 0.1 ml of 0.9% 1 & 2 of 7 daycycle saline (injection grade) 2. HA 0.1 ml containing: 1 & 2 of 7 daycycle 12.5 mg/kg HA 3. 5-FU 0.1 ml containing: 1 & 2 of 7 day cycle 30mg/kg 5-FU 4. HA followed 0.1 ml containing: 1: HA by 5-FU 12.5 mg/kg HAor 2: 5-FU 30 mg/kg 5-FU 3: HA 4: 5-FU of 7 day cycle 5. HA 0.1 mlcontaining: 1: HA 12.5 mg/kg HA 3: HA of 7 day cycle 6. 5-FU 0.1 mlcontaining: 2: HA 30 mg/kg 5-FU 4: HA of 7 day cycle

[0094] Mice were randomly divided into 7 groups of 8 animals per groupfor the short term study and 5 groups of 8 animals for the long termstudy (refer to Table 7 for dosage and treatment administrationschedule).

[0095] The treatment was not extended over the 6 month regimen since ithas been demonstrated that chemotherapy lasting more than six months hasnot generally been associated with greater benefit (Harris et al, 1992).

[0096] Animals were weighed and tumour volumes measured on the day oftreatment application for long term study. In the 6-week study animalswere weighed and tumour volumes measured on a daily basis. Animals wereindividually placed in an injection box, and the injections wereadministered via the tail vein. It has been experimentally proven thatstress can be a major factor in a patients response to chemotherapy(Shackney et al, 1978), therefore we ensured that equal numbers of micewere allocated to each cage, the animal number per cage varied from 5-8depending on the stage of experimentation.

[0097] The experimental end-point occurred when the animal had to beeuthanised due to degree of disease progression or when the 6 month(long term) or 6 week (short term) treatment regimen was completed. Dueto the animal ethics guidelines the animals were monitored fortnightlyby an independent animal ethics officer who assessed the degree ofdisease progression. The following criteria were used to determine if ananimal had reached the stage of experimental end-point of necessarydeath:

[0098] 1). Tumour mass was so large the animal was immobilised;

[0099] 2). Animal was not eating or drinking and had experienceddramatic weight loss; or

[0100] 3). Tumour size was greater than 10% of body mass.

[0101] At the experimental end-point the animals were anaesthetized by a0.1 ml intra-peritoneal injection of Nembutal (60 mg/ml), blood wascollected followed by killing of the animals using cervical dislocation.

[0102] Immediately after killing the mouse the tumour, liver, heart,spleen, bladder, left and right kidneys, uterus, lungs, stomach,intestines, brain and lymph nodes were excised and placed in 4% formalinbuffered with 0.06M phosphate pH 7.5, and cetylpyridinium chloride, 1.0%w/v. The tissue was fixed for 16-24 h before histological processing.Fixed tissue was dehydrated stepwise to 100% ethanol and embedded inparaffin blocks from which 2-4 μm sections were placed on glassmicroscope slides. Staining the tissue sections with a haematoxylinnuclear stain and eosin cytoplasmic stain highlighted any pathologicalfeatures that could indicate treatment toxicity.

[0103] Nine to 11 lymph nodes were collected per animal, ensuring thatall nodes which drained the tumour area were collected. There arecurrently two methods used for the detection of lymph node metastasis

[0104] i) routine haematoxylin and eosin staining of gross organstructure; and

[0105] ii) immunohistochemistry using a cancer marker such ascarcinoembryonic antigen.

[0106] Both methods of metastasis detection were employed in this study.Not all commercially available CEA antibodies react with human breastcancer cells, so we tested the reactivity of 5 different antibodies(DAKO, Amersham and KPL).

[0107] The haematoxylin and eosin stained lymph nodes were examined byDr P. Allen (certified pathologist) where each node was microscopicallyexamined for the presence of tumour cells. The CEA immunostained lymphnodes were microscopically examined, where any positively stained nodeswere counted and considered positive for lymph node metastasis.

[0108] Tumour volume was monitored on a daily or weekly basis bycalliper measurements and tumour volume calculated as previouslydescribed. At the end of the 6 week study, tumour mass was determinedwhere the HA chemosensitizing therapy had significantly smaller tumoursthan the saline group, HA and 5-FU groups (p=0.005) as seen in FIG. 6.No significant differences in tumour response were noted in the initial2 weeks of treatment, but thereafter the HA followed by 5-FU tumourgrowth was retarded in comparison to the other treatment groups. Duringthe 6 weeks of treatment interesting differences were noted in thenumber of tumour doubling cycles. Mice receiving the saline treatmentunderwent an average of 4 tumour doublings, while the incorporation ofHA into the treatment regimen significantly increased the tumourdoubling time where HA/5-FU animals underwent an average of one tumourdoubling cycle, once again highlighting the effect of HA on 5-FUcytotoxicity.

[0109] All animals displayed lymph node metastasis in lymph nodes thatwere adjacent to the primary tumour. The percentage of lymph nodeinvolvement (number of metastatic nodes per animal) was greatly reducedby the HA followed by 5-FU, 5-FU and HA treatment, where the salinegroup demonstrated a 6-fold increase in the amount of lymph nodeinvolvement. The other treatment groups demonstrated a significantlysmaller percentage at 12.2- 14.3% (Dunnett's Multiple Comparison Test,p=<0.001).

[0110] The co-administration of HA resulted in a significant reductionin non-lymphoid metastasis. With the exception of the mice receiving theHA therapy, new tumours were observed around the neck or underarm regionof the area adjacent to the primary tumour.

[0111] Gastro-Intestinal Tract Toxicity:

[0112] One of the most common toxic effects of 5-FU is on thegastro-intestinal tract where haemorrhagic enteritis and intestinalperforation can occur (Martindale, 1993). Animals were monitored dailyfor GI tract upset such as diarrhoea and weekly for more severe toxicitymanifestations such as weight loss. Weight loss was monitored bycalculating net body weight as estimated by subtracting tumour weight,which was calculated as 1 g× tumour volume (cm³) as cited in Shibamotoet al, 1996. For demonstration of any weight changes the animal bodyweight was normalised to the body weight at the time of treatmentcommencement as

Body mass (ex tumour)−body mass at commencement of treatment (extumour)/Body mass at commencement of treatment (ex tumour)×100

[0113] No treatment toxicity was noted throughout the 6-week study. Incomparison to the 5-FU treatment group the mice receiving HA therapy,that is as a sole agent or as a chemosensitizer, demonstrated enhancedwell being where the animal did not loose weight, but maintained itsbody mass (FIG. 4).

[0114] Blood Marrow Suppression

[0115] As one of the major toxicities associated with 5-FU treatment isdepression of the bone marrow and subsequent drop in white blood cellsit was necessary to assess any treatment associated blood toxicity. Uponanaesthetising the animals, blood was collected from the heart or greatvessels using a needle and syringe. Estimation of white blood cellnumber by making a {fraction (1/50)} dilution of blood in mouse tenacitysaline (M) and counting it on a haemocytometer. A differential bloodcount was performed by counting-neutrophils, lymphocytes, anderythrocytes. The total estimation of blood cell sub-populations wascompared to published data for mouse blood.

[0116] The total white cell count and sub-population differential werenot significantly different, regardless of the treatment.

[0117] Effect of Treatment on Organ Mass

[0118] To ensure that treatments did not induce organ atrophy orenlargement, the organs were removed and weighed during the post mortem.The mass of each organ was calculated as a % of the overall net bodyweight, and compared to the organ masses of the saline only group (Group1).

[0119] The overall patient survival time was calculated as the time(days or weeks) that the animal lived after the commencement oftreatment. All animals in each treatment group completed the 6-weektreatment program

[0120] In relation to organ mass, the HA therapy did not result in anydramatic toxicity. Mice receiving 5-FU exhibited an enlarged spleen (61%increase in mass), while the co-administration of HA and 5-FUsignificantly counteracted this enlargement by 31% (student t-test,p<0.001). The 5-FU therapy resulted in a shrinkage of the uterus (22%),once again the HA/5-FU therapy reduced this toxic effect by 10% (studentt-test, p=0.04). It was also clearly defined that the addition of HA tothe treatment regimen, when co-administered or administered the daybefore, significantly decreased the primary tumour mass in comparison tothe saline treatment group (student t-test, p=0.006). No otherdifferences in organ mass were noted between treatments.

EXAMPLE 4 Effect of Hyaluronan Concentration on the In Vitro Efficacy of5-FU

[0121] MDA-MB 468, MDA-MB 435 and MDA-MB 231 cells were cultured asdescribed in Example 2. When the cultures had reached 70-80% confluencythey were washed in 1× HBSS at 37° C. and trypsinised in 10 ml of 0.25%trypsin/0.05% EDTA until cells have fully detached. After add 1 ml ofFCS to neutralise trypsin the cells were counted, centrifuged at 1,200rpm for 5 min and resuspended as follows:

[0122] MDA-MB 231: 12,000 cells/ml of media;

[0123] MDA-MB 468: 25,000 cells/ml of media; and

[0124] MDA-MB 435: 12,000 cells/ml of media.

[0125] Cells were then plated into 48-well plates and incubated inaccordance with suppliers' instructions. After 24 h media was removedand replaced with the following test media:

[0126] MDA-MB 468: 40 nM adriamycin;

[0127] MDA-MB 231: 50 nM adriamycin; and

[0128] MDA-MB 435: 10 nM adriamycin

[0129] 40 nM Adriamycin media:450 ml (Stock adriamycin is 1.7 mM,therefore 1,700,000/40=42,500; 450,000/42500=10.6 ul of 1.7 mMAdriamycin+450 ml Media). Stock HA was 700,000 daltons at 14.3 μM HA

[0130] Conclusions

[0131] This study has definitively proven that HA, can enhance thecytotoxicity of anti-cancer drugs, 5-FU and Adriamycin, both in vitroand in vivo. More specifically:

[0132] 1). As a sole agent HA can exert a cytotoxic effect on cancercells both in vitro and in vivo (FIG. 5);

[0133] 2). Evaluation of the therapeutic efficacy of HA sole therapy orchemosensitizing therapy demonstrated that it was not toxic to normaltissue and it did not enhance the toxicity profile of the drug. In fact,mice receiving the therapy displayed a significant weight gain over the6-week treatment period and a reduction in lymph node metastasis. Theco-administration of HA and 5-FU had a dramatic effect on the reductionof the primary tumour volume; and

[0134] 3). Mice who had HA incorporated into the treatment regimen didnot display the formation of any secondary tumour (FIG. 6).

[0135] Future Studies

[0136] Experiments are presently being conducted on the use of HA for invivo treatment of breast cancer. These experiments are focusing on theeffect of HA concentration and molecular weight and on the cytotoxicityof adriamycin. It is the aim of these studies to also establishing drugand HA exposure time and regimens, as well as the mechanism of action ofHA, ie: receptor mediated transport and/or effect on cell membrane.Further data on the role of HA in chemosensitizing drug-resistant cancercells will also be collected.

[0137] Section 1:

[0138] All studies will be conducted on breast cancer cell lines thatexpress differing levels of HA receptors, CD 44 and RHAMM. Cell lines tobe tested, MDA-MB 435, MDA-MB 231, MDA-MB 468, ZRL-751 and several MDR-1expressing breast cancer cell lines.

[0139] Investigation of the effect of HA/adriamycin exposure times andconcentration on drug-resistant and drug-sensitive breast cancer cells.Four MDR-1 positive and 4 MDR-1 negative cell lines will be exposed toadriamycin at 1, 2.5, 5, 10, 20, 40, 60, 80 and 100 nM, the followingvariables will be tested:

[0140] 1). 1 h drug±100 nM HA exposure followed by 3 days of drug-freegrowth;

[0141] 2). Constant drug exposure±100 nM HA for 3 days 30 min 100 nM HAexposure, followed by drug for 1 h, cells grown drug-free for 3 days;and

[0142] 3). 24 h 100 nM HA exposure, followed by drug for 1 h, cellsgrown drug-free for 3 days.

[0143] These experiments will establish; optimal HA exposure times andregimens, magnitude of increased adriamycin cytotoxicity when combinedwith HA and whether HA can overcome efflux pump resistance in breastcancer cells.

[0144] To date the IC₅₀ of adriamycin has been determined as 90 nM.Using 90 nM of adriamycin the HA (700 kD) concentration will be variedto 1, 3, 10, 30, 100, 300 nM, 1 μM, 3 μM, 10 μM, 30 μM and 100 μM. Theincubation variables to be tested are:

[0145] 1). 30 min HA exposure followed by 1 h drug exposure cells growndrug-free for 3 days;

[0146] 2). 24 HA exposure followed by 1 h drug exposure cells growndrug-free for 3 days; and

[0147] 3). HA±drug exposure for 1 hr, cells grown drug-free for 3 days.

[0148] Any detached cells will be tested for cell viability since it hasbeen suggested that HA can play a pivotal role in cancer cell detachmentand migration. If detached cells are viable the HA receptor status willbe determined using FACS surface epitope identification. Similarexperiments will be performed with short HA oligiosaccharides, ie: 4sacc, 6 sacc, 12 sacc, 5600 Da, 50,000 Da, 100,000 Da, 250,000 Da.

[0149] These experiments will demonstrate the optimal HA:drug ratio invitro, optimal HA exposure time and regimen, effect of HA molecularweight on adriamycin cytotoxicity.

[0150] After determining the optimal HA concentration, the IC₅₀ ofadriamycin will be used in a series of time course experiments toobserve any effect of HA on adriamycin metabolism.

[0151] The [¹⁴C] adriamycin will be exposed to the cells for 30 min, 1h, 2 h, 4 h, 8 h, 16 h and 24 h. The experimental conditions will be:

[0152] 1). Exposure of cells to HA for 30 min followed by drug; and

[0153] 2). Exposure of cells to HA for 24 followed by drug Co-exposureof HA/adriamycin.

[0154] Cells will be removed, hypotonically lysed and centrifuged at113,000 gav for 1 hr. The membrane pellet and supernatant will becounted and analysed for metabolites using HPLC.

[0155] Cells will also be grown on coverslips, where they will beexposed to adriamycin± HA (exposures regimen as above) and a confocalphotography time course will be used to track the cytosolic uptake andmovement of the drug.

[0156] Identification of HA Receptors on MDR-1 positive and negativebreast cancer cell lines, FACS quantitation of the CD44s, CD44v6,CD44v10 and RHAMM receptors will be conducted. Quantitation of theHA/receptor binding and saturation kinetics using FITC/HA and FACSanalysis will also be done.

[0157] By exposing the cells to:

[0158] 1). HA for 30 min followed by drug;

[0159] 2). HA for 24 h followed by drug; and

[0160] 3). HA/adriamycin

[0161] We will be able to determine any of these block CD44s and RHAMMreceptors. The receptor status of any viable cells will be quantitatedusing surface epitope FACS analysis. If blocking of the HA receptorsdecreases the normally observed synergism between adriamycin and HA, themembrane bound and cytosolic adriamycin will be quantited±HA receptorblocking.

[0162] HA degradation by cell lines using [³H]HA and gel filtrationchromatography±receptor blocking will be studied.

[0163] HA of molecular weight, 4 sacc, 6 sacc, 12 sacc, 5600 Da, 50,000Da, 100,000 Da, 250,000 Da, 750,000 Da and 1,500,000 Da will beincubated with breast cancer cell lines at pre-determined“observed-effect” concentrations and the following will be parametersinvestigated: Extracellular and intracellular calcium flux (cellularprobe assays). Regulation of cytoskeletal components (micro-array ofcytoskeletal genes), effect on volume of cells (Coulter size Analysis)and mobility of cancer cells (Boyden Chamber matrigel assays) will alsobe conducted.

[0164] The effect of HA on the cell cycle will be undertaken byincubating HA of molecular weight, 4 sacc, 6 sacc, 12 sacc, 5600 Da,50,000 Da, 100,000 Da, 250,000 Da, 750,000Da and 1,500,000 Da withbreast cancer cell lines at pre-determined “observed-effect”concentrations. Cells will be labelled with potassium iodide andsubjected to FACS analysis. The number of cells in each stage of thecell cycle will be determined.

[0165] Comparisons of the in vitro efficacy of the liposomal Doxorubicinand HA/Doxorubicin preparations will be conducted using the optimalHA/Doxorubicin preparation and the dosage range used by the LiposomeCompany in the pre-clinical testing of the liposomal doxorubicin.

[0166] Section 2:

[0167] Before progression of the HA/adriamycin anti-cancer therapy intoPhase I human breast cancer trials it is necessary to conductpreliminary toxicity experiments. The experiments will focus on:

[0168] 1). Effect of hyaluronan on adriamycin uptake in mouse bodyorgans and fluids;

[0169] 2). Establish a preliminary dose range for adriamycin Determineif HA targets adriamycin to human breast tumour xenografts in nude mice;

[0170] 3). Compare the commercial liposomal Doxorubicin toHA/doxorubicin uptake in mice; and

[0171] 4). Comparison of short-term efficacy of liposomal doxorubicinand HA/doxorubicin.

[0172] From Inaba et al, (1988) the dose of adriamycin in nude mice was4 mg/kg which is a human equivalent dose of 60 mg/m². Nude mice bearinghuman tumours will be injected with adriamycin±HA. Using adraimycinconcentrations of 4 mg/kg±12.5 mg/kg HA. The experimental protocol willinclude the following treatment groups:

[0173] 1). 4 mg/kg adriamycin;

[0174] 2). 4 mg/kg adriamycin+12.5 mg/kg HA; and

[0175] 3). 4 mg/kg liposomal doxorubicin.

[0176] Using adriamycin±HA will be quantitatively injected into the tailvein of the mouse.

[0177] At the time intervals of 2, 15, 30, 60 min and 1.5, 2, 4, 8, 24and 48 h (4 animals/time point) the mice will be killed by a 0.1 ml IPinjection of Nembutal. All body organs, skeletal muscle, lymph nodes,bone marrow, urine and blood will be removed and the adriamycin contentdetermined using HPLC and fluorescence.

[0178] Human breast tumours will be generated in nude mice (WEHI CBAstrain). The mice will be injected with:

[0179] 1). Mouse LD₅₀ is 10 mg/kg;

[0180] 2). 4 mg/kg adriamycin;

[0181] 3). 4 mg/kg adriamycin+12.5 mg/kg HA;

[0182] 4). 8 mg/kg adriamycin;

[0183] 5). 8 mg/kg adriamycin+12.5 mg/kg HA;

[0184] 6). 4 mg/kg liposomal doxorubicin;

[0185] 7). Saline; and

[0186] 8). 12.5 mg/kg HA.

[0187] The above mentioned will be quantitatively injected into the tailvein of the mouse (8 animals/group) on Days 2,4,6 of a weekly cycle.

[0188] Tumour volume, body mass, food intake and functionality of themice will be monitored on a daily basis.

[0189] At the completion of the 8-week study the mice will be killed bya 0.1 ml IP injection of Nembutal. All body organs, tumour, skeletalmuscle, lymph nodes, bone marrow, urine and blood will be removedprocessed for pathological assessment.

[0190] Section 3:

[0191] To answer some basic questions about the effect of HA anti-cancertherapy on colon cancer cells the following experiments should beconducted.

[0192] Investigation of the effect of HA/5-FU exposure times andconcentration on drug-resistant and drug-sensitive colon cancer cells.

[0193] Three resistant and 3 sensitive cell lines will be exposed to5-FU at 1, 2.5, 5, 10, 20, 40, 60, 80 and 100 nM, the followingvariables will be tested:

[0194] 1). 1 h drug±100 nM HA exposure followed by 3 days of drug-freegrowth;

[0195] 2). Constant drug exposure±100 nM HA for 3 days;

[0196] 3). 30 min 100 nM HA exposure, followed by drug for 1 h, cellsgrown drug-free for 3 days; and

[0197] 4). 24 h 100 nM HA exposure, followed by drug for 1 h, cellsgrown drug-free for 3 days.

[0198] Using the IC₅₀ of 5-FU as determined as above, HA (700 kD)concentration will be varied to 1, 3, 10, 30, 100, 300 nM, 1 μM, 3 μM,10 μM, 30 μM and 100 μM. The incubation variables to be tested:

[0199] 1). 30 min HA exposure followed by 1 h drug exposure cells growndrug-free for 3 days;

[0200] 2). 24 HA exposure followed by 1 h drug exposure cells growndrug-free for 3 days; and

[0201] 3). HA±drug exposure for 1 hr, cells grown drug-free for 3 days.

[0202] Any detached cells will be tested for cell viability since it hasbeen suggested that HA can play a pivotal role in cancer cell detachmentand migration. If detached cells are viable the HA receptor status willbe determined using FACS surface epitope identification.

[0203] Similar experiments will be performed with short HAoligiosaccharides, ie: 4 sacc, 6 sacc, 12 sacc, 5600 Da, 50,000 Da,100,000 Da, 250,000 Da.

[0204] After determining the optimal HA concentration, the IC₅₀ of 5-FUwill be used in a series of time course experiments to observe anyeffect of HA on adriamycin metabolism.

[0205] The [³H] 5-FU will be exposed to the cells for 30 min, 1 h, 2 h,4 h, 8 h, 16 h and 24 h. The experimental conditions will be:

[0206] 1). Exposure of cells to HA for 30 min followed by drug; and

[0207] 20. Exposure of cells to HA for 24 followed by drug Co-exposureof HA/5-FU.

[0208] Cells will be removed, hypotonically lysed and centrifuged at113,000 gav for 1 hr. The membrane pellet and supernatant will becounted and analysed for metabolites using HPLC.

[0209] Cells will also be grown on coverslips, where they will beexposed to 5-FU±HA (exposures regimen as above) and a confocalphotography time course will be used to track the cytosolic uptake andmovement of the drug.

[0210] Identification of HA Receptors on resistant and sensitive coloncancer cell lines, FACS quantitation of the CD44s, CD44v6, CD44v10 andRHAMM receptors, Quantitation of HA/receptor binding and saturationkinetics using FITC/HA and FACS analysis will be done.

[0211] Blocking of CD44s and RHAMM receptors with inhibitory antibodies,apply 5-FU±HA following the protocols of:

[0212] 1). Exposure of cells to HA for 30 min followed by drug; and

[0213] 2). Exposure of cells to HA for 24 followed by drug co-exposureof HA/5-FU.

[0214] Cells will be counted. The receptor status of any viable cellswill be quantitated using surface epitope FACS analysis.

[0215] If blocking of the HA receptors decreases the normally observedsynergism between 5-FU and HA, the membrane bound and cytosolic 5-FUwill be quantited±HA receptor blocking.

[0216] HA degradation by cell lines using [³H] HA and gel filtrationchromatography±receptor blocking will be studied.

[0217] Effect of HA on the plasma membrane Hyaluronan of molecularweight, 4 sacc, 6 sacc, 12 sacc, 5600 Da, 50,000 Da, 100,000 Da, 250,000Da, 750,000 Da and 1,500,000 Da will be incubated with breast cancercell lines at pre-determined “observed-effect” concentrations and thefollowing will be parameters investigated:

[0218] 1). Extracellular and intracellular calcium flux (cellular probeassays);

[0219] 2). Regulation of cytoskeletal components (micro-array ofcytoskeletal genes);

[0220] 3). Effect on volume of cells (Coulter size Analysis);

[0221] 4). Mobility of cancer cells (Boyden Chamber matrigel assays);

[0222] 5). Quantitation of HA receptors (FACS); and

[0223] 6). Membrane potential (method to be determined).

[0224] An investigation of the role of HA neo-adjuvant therapy on theinhibition of organ metastasis will be undertaken. In comparison toother treatment groups, mice receiving the HA therapy have demonstratedthat:

[0225] 1). Reduced lymph node metastasis as compared to other treatmentgroups;

[0226] 2). Inhibition of new tumour formation;

[0227] 3). Increased weight gain; and

[0228] 4). Enhanced well-being.

[0229] These results highlight the possible role of HA anti-cancertherapy as an efficient means of reducing the spread of cancer. Throughthe obligatory choice of a pre-clinical model there is a restriction,whereby the spread of the secondary cancer normally occurs in thesurrounding lymph nodes. It would be advantageous to use a model wherewe can examine the spread of the cancer to every organ and the bone. Byusing a model known as the BAG vector metastasis model we would be ableto monitor the spread of cancer to every organ and the bone.

[0230] In brief, the BAG vector consists of a neomycin-resistant LacZgene that can be stably transfected into human breast cancer cells.After intracardiac injections into the nude mice, followed by a 6-weektreatment program it is possible to PCR detect the LacZ gene in anymetastasizing cells/organs. Faxitron scanning with detection of bonelesions would detect any bone metastasis.

[0231] The below treatments will be administered on Day1 , Day 2 of aweekly cycle, for 6 weeks. The treatment groups (5 animals per group)will consist of:

[0232] 1. Saline

[0233] 2. 30 mg/kg 5-FU Day 1, Day 2;

[0234] 3. 12.5 mg/kg HA Day 1, Day 2;

[0235] 4. 30 mg/kg 5-FU+12.5 mg/kg HA (co-administered on Day 1, Day 2);

[0236] 5. 12.5 mg/kg HA on Day 1, 30 mg/kg 5-FU on Day 2, 12.5 mg/kg HAon Day 3, 30 mg/kg 5-FU on Day 4;

[0237] 6. 12.5 mg/kg HA on Day 1,3;

[0238] 7. 30 mg/kg 5-FU on Day 2, 4;

[0239] 8. 15 mg/kg MTX Day 1, Day 2;

[0240] 9. 15 mg/kg MTX+12.5 mg/kg HA (co-administered on Day 1, Day 2);

[0241] 10. 12.5 mg/kg HA on Day 1, 15 mg/kg MTX on Day 2, 12.5 mg/kg HAon Day 3, 15 mg/kg MTX on Day 4;

[0242] 11. 15 mg/kg MTX on Day2, Day 4;

[0243] 12. 15 mg/kg MTX, 30 mg/kg 5-FU, 30 mg/kg cyclophosamide on Day1, Day 2; and

[0244] 13. 15 mg/kg MTX, 30 mg/kg 5-FU, 30 mg/kg cyclophosamide+12.5mg/kg HA on Day 1, Day 2; 12.5 mg/kg HA on Day 1, (15 mg/kg MTX, 30mg/kg 5-FU, 30 mg/kg cyclophosamide) on Day 2, 12.5 mg/kg HA on Day 3,(15 mg/kg MTX, 30 mg/kg 5-FU, 30 mg/kg cyclophosamide) on Day 4.

[0245] Neo-Adjuvant Therapy:

[0246] Immediately before intracardiac injection administer thefollowing:

[0247] 1). 12.5 mg/kg HA;

[0248] 2). 15 mg/kg MTX;

[0249] 3). 15 mg/kg MTX, 12.5 mg/kg HA;

[0250] 4). 30 mg/kg 5-FU;

[0251] 5). 30 mg/kg 5-FU, 12.5 mg/kg HA;

[0252] 6). 15 mg/kg MTX, 30 mg/kg 5-FU, 30 mg/kg cyclophosamide; and

[0253] 7). 15 mg/kg MTX, 30 mg/kg 5-FU, 30 mg/kg cyclophosamide+12.5mg/kg HA.

[0254] Mouse mass and well being will be monitored daily for 6 weeks. Oncompletion of the treatment cycle, each mouse will be scanned for bonelesions. After scanning each organ and body fluid will be removed. Asufficient cross section of the organ will be kept for possible futurepathological analysis, while the remaining tissue will be homogenizedand subjected to competitive PCR for the detection of the LacZ gene.

[0255] Any organs which exhibit metastasis will be histologicallyprocessed and the pattern of colonization of the cancer cells will benoted using galactosidase staining of the Lac Z gene.

[0256] References

[0257] Culty, M., Shizari, M., Erik, W., Thompson. and Underhill, C. B.(1994). Binding and degradation of hyaluronan by human breast cancercell lines expressing different forms of CD44: Correlation with invasivepotential. Journal of Cellular Physiology 160: pp 275-286.

[0258] Culty, M., Nguyen, H A, and Underhill, C B. (1992). Thehyaluronan receptor (CD44) participates in the uptake and degradation ofhyaluronan. J Cell Biol 116 (4): pp1055-1062.

[0259] Lang F., Ritter M., Volkl H and Haussinger D (1993). Thebiological Significance of cell volume Ren Physiol Biochem. 16: pp.48-65.

[0260] Wang, C., Zhang, S. and Turley, EA. (1996). The role ofhyaluronan and hyaluronan receptors in breast cancer cell invasion,motility and proliferation. In: Fourth International Workshop onHyaluronan in Drug Delivery. (Editor: Willoughby, D. A)Roy.Soc.Med.Press. pp 37-53.

[0261] Wang, C., Tammi, M., Guo, H. and Tammi, R. (1997). Hyaluronandistribution in the normal epithelium of esophagus, stomach, and colonand their cancers. American Journal of Pathology. 148 (6): pp 1861-1869.

The claims defining the invention are as follows:
 1. A method oftreating a drug resistant disease in a subject in need thereofcomprising the step of administering to said subject a therapeuticallyeffective amount of hyaluronan in conjunction with a chemotherapeuticagent such that said chemotherapeutic agent is more effective than whenadministered alone.
 2. A method of enhancing the bioavailability of achemotherapeutic agent comprising the step of administering to a subjectin need thereof a therapeutically effective amount of hyaluronan.
 3. Amethod of treating or preventing multidrug resistance or drug-resistantcells comprising the step of administering a sufficient amount ofhyaluronan, prior to, together with, or subsequent to the administrationof a chemotherapeutic agent.
 4. A method according to any one of claims1 to 3, wherein the chemotherapeutic agent is selected from the groupconsisting of carmustine (BCNU), chlorambucil (Leukeran), cisplatin(Platinol), Cytarabine, doxorubicin (Adriamycin), fluorouracil (5-FU),methoxetrate (Mexate), CPT111, etoposide, plicamycin (Mithracin) andtaxanes.
 5. A method according to claim 1, wherein the drug resistantdisease is a cellular proliferative disorder.
 6. A method according toclaim 5, wherein the cellular proliferative disorder is selected fromthe group consisting of cancers of the breast, lung, prostate, kidney,skin, neural, ovary, uterus, liver, pancreas, epithelial, gastric,intestinal, exocrine, endocrine, lymphatic, hematopoietic system or headand neck tissue.
 7. A method according to any one of claims 1 to 6,wherein the subject is mammal.
 8. A method according to claim 7, whereinthe mammal is selected from the group consisting of bovine, canine,equine, feline, porcine and human.
 9. A method according to any one ofclaims 1 to 8, wherein the administration of hyaluronan, is prior to,together with, or subsequent to the administration of a chemotherapeuticagent.
 10. A method according to any one of claims 1 to 9, wherein theadministration of hyaluronan and/or chemotherapeutic agent is orally,topically, or parenterally.
 11. A method according to claim 10, whereinthe hyaluronan and/or chemotherapeutic agent is administered togetherwith a pharmaceutically acceptable carrier, adjuvant, or vehicle.
 12. Amethod according to claim 10 or claim 11, wherein parenteraladministration is either by subcutaneous injection, aerosol,intravenous, intramuscular, intrathecal, intracranial, intrasternalinjection or infusion techniques.